Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii.
Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monoclonal antibodies to screen a cDNA expression library in lambda gt11, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18,959. As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate.
Prince
J B
JB
Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, CA 94301.
Auer
K L
KL
Huskinson
J
J
Parmley
S F
SF
Araujo
F G
FG
Remington
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015183:Restriction Mapping; D014122:Toxoplasma / Q000235:genetics* / Q000276:immunology
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C067547:surface antigen P22, Toxoplasma; D004247:DNA